Objective To establish a flowcytometry method for detecting phagocysis of Mycobacterium tuberculosis(Mth) by the human peripheral blood neutrophils( PMNs), and to explore the effects of Thl and Th2 cytokine on phagocytotic activity of PMNs. Methods By using acid-fast staining the phagocytosis of Mtb by human neutrophil was observed by microscopy, and the phagocytosis of FTTC labeled Mtb by human neutrophils was detected under confocal microscope. The whole fresh peripheral blood of healthy adults was incubated with FTTC labeled Mtb and the phagocytosis were measured by flow cytometry. The altered phagocytosis of FTTC-Mtb by neutrophils pretreated with IL-2, IFN-γ, GM-CSF or IL-4 were measured. Results Human peripheral blood neutrophils activity of Mtb phagocytosis was observed by acid-fast staining and confocal microscope. The percentage of phagocytosis of Mtb was dependent on the time of incubation with Mtb. The percentages were 47% at 5 min and reached plateau about 66% ～72% at 15 min to 20 min.Pretreatment with different concentrations of IL-2 or IFN-γincreased the phagocytosis of Mtb by 76.7% and 75.2%, respectively; but pretreatment with IL-4 decreased the phagocytosis by 31.7%. Conclusion IL-2and IFN-γcan increase phagocytosis of Mth by neutrophils; while IL-4 can reduced neutrophil activity of phagocytosis of Mtb by human neutrophils, and demonstrate that Th1/Th2 type cytokins involve in regulating the acitvities of neutrophils anti-Mtb infection.

To observe the effect of Mycobacterium tuberculosis 19 kDa lipoprotein (Mtb P19) upon the expression and distribution of Toll-like receptor-2 (TLR-2) on the surface of macrophages, Mtb P19 was prepared from the cultured M.tuberculosis H37 Ra strain , and phorbol myristatye acetate (PMA)-differentiated THP-1 cells were co-cultivated with Mtb P19 at concentration of 10 g/mL and at 37 ℃ temperature and a condition containing 5% CO_2.for 6 hours.. The distribution of TLP-2 on the surface of macrophages was investigated by immuno-cellular chemical method (ICC) while the effect of Mtb P19 upon the expression of TLR on the surface of macrophages was assayed by fluorescent antibody staining. In addition,the changes of TLR-2 expression before and after the effect of Mtb P19 were investigated by flow cytometry analysis and change of TLR-2 arrangement after stimulation with Mth P19 was determined by co-focal microscopy. It was found that the TLR-2 molecules were evenly distributed on the surface of macrophages as demonstrated by ICC. The mean fluorescent intensity increased significantly after stimulation with Mtb P19 for 6 hours in comparison with that of the control group, and the patchy surface with fluorescent staining positive zones could be detected on the surface of macrophages. Nevertheless , the distribution of TLR-2 molecule in the control group appeared to be randomly dynamic. It is evident that the Mth P19 not only can induce the surface expression of TLR-2 molecules, but also cause a functional aggregation of this receptor.

To observe the influence of Mycobacterium tuberculosis 19-kDa lipoprotein (Mtb P19) on function and phenotype of macrophage,the Mtb P19 was prepared from cultured Mtb H37Ra and the phorbol myristate acetate-differentiated THP-1 cells were incubated with P19 at the concentration of 10 μg/mL with 5% CO_2 at 37℃ for up to 48 hours.Supernatants were collected for TNF-α and IL-6 detection by ELISA,then the phenotype fluorescent antibodies were stained to analyze HLA-DR expression changes between control group and experimental group.Flow cytometry and microscopy was used to assay the phagocytosis in macrophages stimulated by Mtb P19.IL-6 and TNF-α in collected supernatants were detected.Results indicated that both were found significant increases and their phagocytosis were enhanced.Comparing to the control group,the mean fluorescence intensity showed a significant increase 24hs after stimulation.It presents that Mtb P19 could be able to induce macrophages activation,and it would be significantly important for protection during infection period.